The separation and/or detection of ionic species is generally carried out by utilizing electrochemical properties of analytes, such as ionic interactions and conductivity in ion chromatography or ionic mobility in capillary electrophoresis. Ion chromatography (IC) is capable of detecting simultaneously a large variety of ionic species at low concentration levels. The ability to separate and detect several ionic species simultaneously is a unique characteristic of IC. However, there are important limitations to IC, including lack of sufficient selectivity for certain types of mixtures, low separation efficiency and a relative complexity of instrumentation.
Capillary electrophoresis (CE) is an efficient analytical separation technique for analysis of minute amounts of sample CE separations are performed in a narrow diameter capillary tube, which is filled with an electrically conductive medium termed the "carrier electrolyte". A current is applied to the carrier electrolyte, and ionic species in the sample move from one electrode toward the other at a rate which is dependent upon certain characteristics, such as molecular charge, size and/or mobility CE may be performed using gels or liquids, such as buffers, in the capillary. In the liquid mode, known as free zone electrophoresis, separations are based on the ratio of charge to Stoke's radius.
CE has several advantages over IC and conventional gel electrophoresis for the separation of ionic species. These include faster separation speed, improved resolution and smaller sample size. For example, separation speeds using CE can be 10 to 20 times faster than conventional gel electrophoresis, and no post-run staining is necessary. In part, high resolution can be obtained through the use of high voltages because of the rapid dissipation of heat by the capillary. Further, band broadening is minimized due to the narrow capillary diameter. In free-zone electrophoresis, the phenomenon of electroosmosis, or electroosmotic flow (EOF), which is the bulk flow of liquid rapidly moves all of the sample molecules whether they are positively charged, negatively charged or neutral. Under certain conditions EOF can contribute to improved resolution and separation speed in free-zone CE.
The detection of some ionic species by CE is problematical, however, due to the "transparency" of many ionic species to light. These ions do not absorb light, so they cannot be detected by conventional photometric means, e g., direct photometric or fluorescent detection. However, these ions can be detected using indirect photometric detection. Indirect photometric detection relies upon the presence of a light absorbing electrolyte ion in the background electrolyte. Non-absorbing species are detected as zones of decreased absorbance or voids in the background due to the displacement of the light absorbing electrolyte ion. Indirect photometric detection has been described using fluorescent, ultraviolet (UV) and UV-visible (UV-vis) absorbing ions in the background electrolyte. For example, Small et al. in U.S. Pat. No. 4,414,842 describe a technique for detecting ions in an ion exchange chromatography system by indirect UV detection in which a UV-absorbing ion is included in the elution buffer. Other methods utilizing indirect photometric detection in chromatography have been described by Foret et al., J. Chromatography, 470:299-308 (1989); Kuhr et al., Anal. Chem., 60:2642-2646 (1988); Kuhr et al., Anal. Chem., 60:1832-1834; and Takeuchi et al., Chromatographia, 25:1072-1074 (1988). The need exists for a method for separating and detecting ionic molecules which is faster, more efficient, has better resolution, and requires less sample preparation than the available methods.